Method for the isolation of novel antigens

Abstract

Methods for isolating novel antigens that stimulate CD4-positive T cells are disclosed. The methods provided comprise the steps of: transforming a host cell with a plasmid suspected of containing such a DNA sequence encoding such an antigen and inducing antigen expression; incubating the transformed host cell with at least one dendritic cell for a period of time sufficient to form a peptide/MHC Class II complex on the dendritic cell, the peptide being derived from the expressed antigen; incubating the dendritic cell with CD4+ T cells; determining the level of CD4+ T cell stimulation, thereby identifying a transformed host cell that expresses at least one CD4+ T cell-stimulating antigen; and isolating DNA from the transformed host cell. DNA sequences isolated using such methods, together with antigens encoded by such DNA sequences, are also provided.

1 . A method for identifying DNA sequences that encode CD4+ T cell stimulating antigens, comprising: 
 a) transforming a host cell with a plasmid suspected of containing a DNA sequence that encodes a CD4+ T cell stimulating antigen and inducing antigen expression;    b) incubating the transformed host cell with at least one dendritic cell for a period of time sufficient to form an MHC II/peptide complex on the dendritic cell, wherein the peptide is derived from the expressed antigen;    c) incubating the dendritic cell with CD4+ T cells and determining the level of CD4+ T cell stimulation, thereby identifying a transformed host cell that expresses at least one CD4+ T cell stimulating antigen; and    d) isolating DNA from the transformed host cell.    
     
     
         2 . The method of  claim 1  wherein the level of CD4+ T cell stimulation is determined by measuring production of at least one cytokine.  
     
     
         3 . The method of  claim 2  wherein the cytokine is interferon-gamma.  
     
     
         4 . The method of  claim 1  wherein the level of CD4+ T cell stimulation is determined by measuring cell proliferation.  
     
     
         5 . The method of  claim 1  wherein the plasmid contains DNA isolated from an infectious disease agent.  
     
     
         6 . The method of  claim 5  wherein the infectious disease agent is  M. tuberculosis.    
     
     
         7 . The method of  claim 1  wherein the plasmid contains DNA isolated from tumor tissue.  
     
     
         8 . The method of  claim 1  wherein the host cell is selected from the group consisting of  E. coli,  yeast and mammalian cells.  
     
     
         9 . A method for identifying DNA sequences that encode CD4+ T cell stimulating antigens, comprising: 
 a) transforming a host cell with a plasmid suspected of containing a DNA sequence that encodes a CD4+ T cell stimulating antigen and inducing antigen expression;    b) incubating the transformed host cell with at least one dendritic cell for a period of time sufficient to form an MHC II/peptide complex on the dendritic cell, wherein the peptide is derived from the expressed antigen; and    c) incubating the dendritic cell with CD4+ T cells and determining the level of CD4+ T cell stimulation, thereby identifying a transformed host cell that expresses at least one CD4+ T cell stimulating antigen.    
     
     
         10 . The method of  claim 9  additionally comprising isolating DNA from the transformed host cell.  
     
     
         11 . An isolated DNA sequence selected from the group consisting of: 
 a) DNA sequences isolated using the method of  claim 1;     b) DNA sequences complementary to DNA sequences of a); and    c) DNA sequences that hybridize to a DNA sequence of a) or b) under moderately stringent conditions.    
     
     
         12 . An expression vector comprising a DNA molecule according to  claim 11 .  
     
     
         13 . A host cell transformed with an expression vector according to  claim 12 .  
     
     
         14 . A polypeptide comprising an immunogenic portion of an antigen, or a variant of said antigen that differs only in conservative substitutions and/or modifications, the antigen comprising an amino acid sequence encoded by a DNA sequence according to  claim 11 .  
     
     
         15 . A method for identifying DNA sequences that encode antigens comprising antibody epitopes, the method comprising: 
 a) transforming a host cell with a plasmid suspected of containing a DNA sequence that encodes a CD4+ T cell stimulating antigen and inducing antigen expression;    b) incubating the transformed host cell with at least one dendritic cell for a period of time sufficient to form an MHC II/peptide complex on the dendritic cell, wherein the peptide is derived from the expressed antigen;    c) incubating the dendritic cell with CD4+ T cells and determining the level of CD4+ T cell stimulation, thereby identifying a transformed host cell that expresses at least one CD4+ T cell stimulating antigen; and    d) isolating DNA from the transformed host cell.    
     
     
         16 . The method of claim  51  wherein the level of CD4+ T cell stimulation is determined by measuring production of at least one cytokine.  
     
     
         17 . The method of  claim 16  wherein the cytokine is interferon-gamma.  
     
     
         18 . The method of  claim 15  wherein the level of CD4+ T cell stimulation is determined by measuring cell proliferation.  
     
     
         19 . The method of  claim 15  wherein the plasmid contains DNA isolated from an infectious disease agent.  
     
     
         20 . The method of  claim 19  wherein the infectious disease agent is  M. tuberculosis.